s2fieldsofstudy: Biology Medicine

text:
Intrarenal Arterial Transplantation of Dexmedetomidine Preconditioning Adipose Stem-Cell-Derived Microvesicles Confers Further Therapeutic Potential to Attenuate Renal Ischemia/Reperfusion Injury through miR-122-5p/Erythropoietin/Apoptosis Axis

Intravenous adipose mesenchymal stem cells (ADSCs) attenuate renal ischemia/reperfusion (IR) injury but with major drawbacks, including the lack of a specific homing effect after systemic infusion, cell trapping in the lung, and early cell death in the damaged microenvironment. We examined whether intrarenal arterial transplantation of dexmedetomidine (DEX) preconditioning ADSC-derived microvesicles (DEX-MVs) could promote further therapeutic potential to reduce renal IR injury. We evaluated the effect of DEX-MVs on NRK-52E cells migration, hypoxia/reoxygenation (H/R)-induced cell death, and reactive oxygen species (ROS) amount and renal IR model in rats. IR was established by bilateral 45 min ischemia followed by 4 h reperfusion. Intrarenal MVs or DEX-MVs were administered prior to ischemia. Renal oxidative stress, hemodynamics and function, western blot, immunohistochemistry, and tubular injury scores were determined. The miR-122-5p expression in kidneys was analyzed using microarrays and quantitative RT-PCR and its action target was predicted by TargetScan. DEX-MVs were more efficient than MVs to increase migration capability and to further decrease H/R-induced cell death and ROS level in NRK-52E cells. Consistently, DEX-MVs were better than MV in increasing CD44 expression, improving IR-depressed renal hemodynamics and renal erythropoietin expression, inhibiting IR-enhanced renal ROS level, tubular injury score, miR-122-5p expression, pNF-κB expression, Bax/caspase 3/poly(ADP-ribose) polymerase (PARP)-mediated apoptosis, blood urea nitrogen, and creatinine levels. The use of NRK-52E cells confirmed that miR-122-5p mimic via inhibiting erythropoietin expression exacerbated Bax-mediated apoptosis, whereas miR-122-5p inhibitor via upregulating erythropoietin and Bcl-2 expression reduced apoptosis. In summary, intrarenal arterial DEX-MV conferred further therapeutic potential to reduce renal IR injury through the miR-122-5p/erythropoietin/apoptosis axis.

Introduction
Renal ischemia/reperfusion (IR) injury can be caused by cardiac surgery, renal vascular obstruction, and kidney transplantation, mainly leading to acute kidney injury, which is complicated by a lack of effective preventative and therapeutic strategies. Overproduction of reactive oxygen species (ROS) can evoke oxidative stress and trigger the translocation of nuclear factor-kappa B (NF-κB) to the nucleus to activate several inflammatory cytokines and adhesion molecules, leading to inflammation and apoptosis [1]. The increased ROS by IR can enhance oxidative stress signaling, especially Bax/Bcl-2/caspase 3/poly-(ADPribose)-polymerase (PARP)-mediated apoptotic cell death [2], consequently leading to acute renal injury.
Mesenchymal stem cells can be obtained from umbilical cord, placenta, bone marrow, and adipose tissue [3]. Among them, adipose-derived stem cells (ADSCs) are abundant and easy to access vs. stem cells from other sources. The cell-based mesenchymal stem cells have been applied in the clinical trial but with some limitations and challenges, including the cell trapping in the pulmonary microcirculation with clotting risk, the extremely low survival rate of transplanted stem cells in the ischemic tissue, and the unguided homing effect. It is mentioned that ADSCs through paracrine mechanisms provide excellent self-renewal and proliferation ability through high levels of growth factors, cytokines, microvesicles (MVs) or exosomes carrying noncoding RNAs such as microRNAs (miRNAs) in the microenvironment or conditioned medium [4,5]. MiRNAs are small noncoding RNAs with 22~25 nucleotides in eukaryotes that can be contained in MVs and transferred to neighboring or distant cells, and can modulate the function of recipient cells. MiRNAs combined with the 3 -terminal nontranslation region (3 -UTR) of mRNA of the target gene to degrade mRNA or inhibit mRNA translation, to regulate the expression of the target gene to participate in cell survival and proliferation and regulation of various biological processes such as apoptosis, oxidative stress, and other biological processes [6][7][8][9]. MV and exosomes derived from stem cells can facilitate intercellular communication by transferring proteins and miRNAs [6]. The contribution of miRNAs to the pathogenesis of several diseases has been demonstrated previously [7][8][9]. The decreased miR-122 attenuated cisplatin-induced acute kidney injury [7] and cerebral IR injury [9]. Thus, we suggest that ADSC-derived MVs may ameliorate renal IR injury through the action of miRNAs.
A previous report using exendin-4-assisted ADSC therapy was superior to either one alone for preserving kidney function in acute kidney IR injury, suggesting that pharmacological preconditioning ADSCs further enhanced renal protection [10]. Intravenous anesthetics such as dexmedetomidine (DEX) could bind with the α 2 adrenoceptor to exert sedative effects in various clinical situations in intensive care units. In addition, DEX with anti-inflammatory and renoprotective effects [11] and co-treatment with mesenchymal stem cells can further attenuate hepatic IR injury [12]. To date, there is no literature exploring whether DEX-treated ADSC could elevate the efficacy against renal IR injury through DEX preconditioning ADSC-MVs. In the present study, we preconditioned the ADSCs with DEX to explore the DEX-MVs' effect on migration activity, hypoxia/reoxygenation (H/R)-induced renal proximal tubular cells injury, and IR-induced acute kidney injury.

Animals and ADSC Preparation
Female Wistar rats (200 to 250 g) at the age of 10 weeks were purchased from Bio-LASCO Taiwan Co., Ltd. (Ilan, Taiwan) and housed at the Experimental Animal Center, National Taiwan Normal University, at a constant temperature and with a consistent light cycle (light from 07:00 to 18:00 O'clock). Food and water were provided ad libitum. All surgical and experimental procedures were approved by National Taiwan Normal University Institutional Animal Care and Use Committee (Approval number 105035) and were in accordance with the guidelines of the National Science Council of Republic of China (NSC 1997).
Antioxidants 2022, 11, 1702 3 of 23 ADSC were isolated from subcutaneous flank adipose tissue of 10 anesthetized rats as previously described [13,14]. Briefly, under urethane anesthesia, isolated flank adipose tissue was collected into centrifuge tubes under the sterile condition. The adipose tissue was stirred to aspirate off saline and oil phases and washed 3 to 5 times with phosphate buffer saline (PBS), and the lower phase was discarded until clear. The final upper phase was collected and collagenase was added and incubated for 1 to 4 h at 37 • C on a shaker. After incubation, 10% fetal bovine serum (FBS; Invitrogen) was added into the tube to neutralize collagenase. The fluid in the tube containing digested fat was centrifuged at 800× g for 10 min. Then, we aspirated the supernatant composed of floating adipocytes, lipids and liquid, and left stromal vascular fraction (SVF) pellet. We used 160 mM NH 4 Cl to suspend SVF pellet, which was then incubated for 10 min at room temperature. After incubation, the pellet was centrifuged at 400× g for 10 min at room temperature. The final pelleted fraction of mononuclear cells was then resuspended in DMEM (Dulbecco's Modified Eagle's Medium; Sigma-Aldrich, Darmstadt, Germany) supplemented with 40% FBS, penicillin-streptomycin, and 10 ng/mL EGF (Invitrogen) incubated on Petri dish overnight to select for adherent cells. The remaining cells and debris were aspirated off on the next day and the plate was washed with PBS. ADSCs were maintained on DMEM low glucose supplemented with 10% FBS, 1% penicillin-streptomycin and L-glutamine at 5% CO 2 37 • C. Primary ADSC were seeded into a T75 culture flask and grown in complete culture medium DMEM, supplemented with 10% FBS, 100 units/mL penicillin, 100 µg/mL streptomycin, and 2 mM L-glutamine, and changed every 2 days.

Identification of ADSCs by Surface Marker
Isolated ADSCs were seeded into a T75 culture flask for 24 h, then fixed by 4% paraformaldehyde for 30 min and incubated with positive surface marker, FITC mouse anti-rat CD90 (BD Pharmingen, La Jolla, CA, USA), anti-rat CD29 (BD Pharmingen), and a negative marker, FITC mouse anti-rat CD31 (BD Pharmingen), anti-rat CD45 (BD Pharmingen) for 30 min. ADSCs were washed by phosphate-buffered saline (PBS) three times and analyzed by flow cytometry (BD FACSAria III; BD Biosciences, San Jose, CA, USA). A purified ADSC was characterized for highly positive expression of CD44, CD29, and CD90 and negative CD31 and CD45 expression [15].

Purification of MVs
The ADSCs were pretreated with a selective agonist of the α2-adrenergic receptor and imidazoline receptors dexmedetomidine (DEX) [16,17] for 24 h. ADSCs or DEX-treated ADSCs were seeded in 6-well plates for 24 h before being collected in 1.5 mL tube. The tubes with ADSCs were placed in a thermomixer (ThermoMixer ® C, Eppendorf, Germany) for 30 min at 300 rpm room temperature. The isolation procedure of MV was described previously [18,19]. ADSCs were centrifuged 10 min at 300× g, 4 • C to discard dead cells and cell debris. The supernatant was collected and centrifuged three times, which were 10 min 2000× g, 30 min 10,000× g, and 30 min 10,000× g at 4 • C. The final pellets filled with isolated MVs were suspended with PBS and recentrifuged for 30 min at 10,000× g, 4 • C to remove contaminating proteins. We used a scanning electron microscope (JEOL-6500F, JEOL, Tokyo, Japan) to characterize the morphology and size of the MV. The images were analyzed using the ImageJ software (National Institutes of Health, NIH, Bethesda, MD, USA). One previous study indicated that intravenously injected stem cells were trapped in lung tissue and did not reach the damaged kidney tissue [20]. To prevent ADSCs being trapped in lung tissue and to confer high protective efficacy to the damaged kidney, intrarenal arterial MVs or DEX-MVs were administered at the dose of 15 µg/mL in saline. The isolated MV marker was characterized with CD44 by Western blot analysis.

Wound Healing Assay
Wound healing assay was performed to detect the cell migration of NRK-52E cells, which were seeded into a 6-well plate with 1 × 10 5 cells/well for 12 h, and then incubated with MV or DEX-MV for 24 h and grown until 90% confluence. Then, NRK-52E cells created an artificial wound by disposable 10 µL pipette tips. Detaching NRK-52E cells were washed out by serum-free condition medium three times after being scratched and cultured with serum-free condition medium. The NRK-52E cells' migration was imaged under an inverted microscope and photographed at the times of 0 and 24 h of culturing.

Extraction of RNA for miRNA Assay
Extraction of total RNA, small RNA (fewer than 200 nucleotides), and large RNA from MV, DEX-MV, control kidney, IR kidney, and MV-and DEX-MV-treated kidney was performed using a SeraMir TM Exosome RNA Amplification Kit (System bioscience) according to the manufacturer's instructions. The following miRNA was used: rno-miR-122-5p MIMAT0000827 UGGAGUGUGACAAUGGUGUUUG.

Grouping and Renal IR Induction
Twenty-four female rats (n = 6 in each group) were divided into control (CON), ischemia/reperfusion (IR), IR with ADSC-derived MV treatment (IR+MV) and IR with DEX-MV treatment (IR + DEXMV) groups. The rats with or without renal IR (n = 6 in each group) were anesthetized with intraperitoneal urethane (1.2 g/kg). The trachea was exposed via a midline cervical incision and intubated. For the induction of ischemia, the bilateral renal arteries were clamped for 45 min with a small vascular clamp. Shamoperated animals underwent similar operative procedures without occlusion of the renal arteries. Reperfusion was initiated by the removal of the clamp for 4 h. After IR insults, arterial blood and urine were collected for renal functional determination [blood urea nitrogen (BUN) and creatinine]. After sacrifice with intravenous KCl, these two kidneys were resected and divided into two parts. One part was stored in 10% neutral buffered formalin for immunohistochemistry and in situ pathologic assay, and the other was quickly frozen in liquid nitrogen and stored at −70 • C for protein isolation.

Tracking of Intrarenal Arterial Administered MV and DEX MV in Rat Kidneys
To determine the MV or DEX MV expression in the kidney, we injected MV or DEX MV containing a green fluorescent protein (GFP) into the kidney and examined the GFP expression in rat kidneys one hour later. The renal sections were investigated under a fluorescent microscope for the detection of fluorescence in the kidneys. Western blot with primary antibodies against CD44 (MCA643GA, Serotec, Kidlington, UK) was examined in the kidney homogenates for quantification of CD44 expression, which is a positive ADSC surface biomarker [15] in these kidneys.
The in vitro ROS measurement from the NRK-52E cells with 1 × 10 5 cells/well was performed with an ultrasensitive chemiluminescence analyzer, as described previously [1]. Immediately before CL measurement, 0.1 mL of phosphate-buffered saline (pH 7.4) was added to 0.2 mL of cell cultures. The CL was measured in a completely dark chamber of the Chemiluminescence Analyzing System. After 100 s background level determination, 1.0 mL of 0.1 mM lucigenin in phosphate-buffered saline (pH 7.4) was injected into the sample. The CL was monitored continuously for an additional 600 s. The total amount of CL was calculated by integrating of the area under the curve and subtracting it from the background level. The assay was performed in duplicate for each sample and was expressed as CL counts/10 s. The mean ± SEM of the CL level of each sample was calculated.

Renal Microcirculation Determination
To examine the MV and DEX-MV effect on IR-evoked renal hemodynamics change, a full-field laser perfusion imager (MoorFLPI, Moor Instruments Ltd., Devon, UK) was used to continuously quantitate the renal microcirculatory blood flow intensity during the periods of baseline, ischemia, and reperfusion, as described previously [21].

Renal Arterial Blood Flow Determination
To examine MV and DEX-MV effect on IR induced renal hemodynamics alteration continuously, including 60 min of baseline, 45 min of ischemia, and 4 h of reperfusion periods, we measured the renal arterial blood flow by placing the renal artery on a blood flow probe and connected to a transonic flow meter (T420, Transonic System Inc., Ithaca, NY, USA) and recorded on a Power Lab system (ML870 PowerLab 8/30, ADInstruments Pty Ltd., Castle Hill, NSW, Australia).

Histologic Studies
Kidney samples were fixed in 4% paraformaldehyde and embedded in paraffin. Fivemicrometer sections were stained with hematoxylin and eosin (H&E). Twenty sections in each kidney were randomly selected at ×200 magnification, and the degree of renal damage was scored using the scoring system for renal injury, as described previously [20]. The percentage of tubular injury parameters containing epithelial flattening, tubular atrophy, tubular dilatation, and brush border loss was estimated by a five-point scale. The degree of injury was graded on a scale from 0 to 4, where 0 = normal; 1 = mild, involvement of less than 25% of the cortex; 2 = moderate, involvement of 25-50% of the cortex; 3 = severe, involvement of 50-75% of the cortex; and 4 = extensive damage, involving more than 75% of the cortex.

Immunohistochemistry
Five-micrometer tissue sections were deparaffinized in xylene, rehydrated in an ethanol series, and submitted to antigen retrieval step. The sodium citrate buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0) was used for heat-induced epitope retrieval. After 15 min of antigen retrieval step, the sections were blocked for nonspecific binding with 5% bovine serum albumin (Sigma-Aldrich, St. Louis, MO, USA) for 1 h at room temperature and incubated with the primary antibodies for 18 h at 4 • C. These sections were washed with PBS three times and then were incubated with primary antibodies, including rabbit anti-Caspase 3 (1:100; Chemicon), rabbit anti-PARP (1:100, Promega), and rabbit anti-NF-κB (1:100; R&D Systems, Minneapolis, MN, USA).

Luciferase Reporter Assay
The binding sites of EPO and miR-122-5p were predicted using the Targetscan database (http://www.targetscan.org/vert_72/, accessed on 15 April 2021). After transfection, the cells were collected and lysed, followed by detection of luciferase activity with the help of Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA).

Statistical Analysis
All values were expressed as mean ± standard error mean (SEM). Differences within and among groups were evaluated by a one-way analysis of variance with a post hoc comparison. Differences were regarded as significant if p < 0.05 was adapted.

Characteristics and Effect of MV and DEXMV on Cell Viability, Wound Healing Rate, and Antioxidant Activity
We first characterized the isolated ADSCs of cultured cells of the fourth passage by flow cytometry. Our data revealed that 94.9% of cells expressed CD90 positive expression and 99.7% expressed CD45 negative expression ( Figure 1A). We compared the preconditioning effect of DEX to the ADSCs on morphologic analysis. The morphology of ADSC in control and DEX treatment displayed a similar shape in the ADSCs ( Figure 1B), implying a nondetrimental effect of DEX on ADSC culture. We next characterized the particle size of ADSC-derived MVs under a scanning electron microscope and found that the MVs displayed a uniform morphology and size around 58.6 ± 6.1 nm ( Figure 1C). We evaluated the effect of MVs or DEX-MVs in response to hypoxia-/reoxygenation-induced injury in NRK-52E cells by MTT assay. Our data found that H/R reduced NRK-52E cell viability and MVs or DEX-MVs restored the decreased cell viability ( Figure 1D). Furthermore, DEX-MVs were more efficient than MVs in recovering cell viability. The scratch size for evaluating wound healing assay was demonstrated in different time courses between MV or DEX-MV groups ( Figure 1E). DEX-MV treatment showed a higher migration capability compared to MV treatment. In addition, DEX-MVs acted more effectively than MV-and H/R-induced ROS amounts in NRK-52E cells ( Figure 1F).

DEXMVs Were More Efficient Than MVs in Increasing CD44 Expression in Rat Kidneys
We administered the PBS, MVs, or DEXMVs through the intrarenal arterial catheter from the femoral artery to the kidneys, as the graph demonstrates in Figure 2A. To ascertain the localization of MVs in the kidneys, we labeled the MVs with fluorescent dye GFP and identified their localizations in the treated kidneys. Our data showed that the fluorescent MVs indicated by the yellow arrow were found in IRMV and IRDEXMV kidneys but not in CON or IR kidneys ( Figure 2B). The fluorescent intensity of MVs was stronger in IRDEXMV than that in IRMV kidneys ( Figure 2E). We confirmed the CD44 expression, which was a specific surface marker of ADSC-derived MVs, in the kidneys by Western blot ( Figure 2C). We found that CD44 expression was significantly enhanced in IRMV and IRDEXMV kidneys, whereas CD44 expression somewhat decreased in IR kidneys compared to CON kidneys ( Figure 2D). Our result observed that the renal trafficking CD44 expression was much higher in IRDEXMV than that in IRMV kidneys ( Figure 2D), suggesting a higher homing effect in DEXMVs.

MVs and DEXMVs Preserved Renal Microcirculation, Hemodynamics, and Function
To determine the effect of MVs and DEXMVs on kidney ROS, renal microcirculation, renal arterial blood flow, and renal function, we compared these parameters in response to renal IR injury in the rats. As shown in Figure 3A, the in vivo kidney ROS amount was significantly increased in response to IR injury in IR, IR + MV, and IR + DEXMV groups. The IR-enhanced kidney ROS level was significantly depressed by MV or DEXMV treatment ( Figure 3B). Our data further demonstrated that DEXMVs were more efficient than MVs to reduce kidney ROS value.  IRDEXMV than that in IRMV kidneys ( Figure 2E). We confirmed the CD44 expression, which was a specific surface marker of ADSC-derived MVs, in the kidneys by Western blot ( Figure 2C). We found that CD44 expression was significantly enhanced in IRMV and IRDEXMV kidneys, whereas CD44 expression somewhat decreased in IR kidneys compared to CON kidneys ( Figure 2D). Our result observed that the renal trafficking CD44 expression was much higher in IRDEXMV than that in IRMV kidneys ( Figure 2D), suggesting a higher homing effect in DEXMVs.   Figure 3. In response to renal IR injury, in vivo kidney ROS measurement was indicated in four groups of rats (n = 6 each) (A). The average ROS amount (n = 6 each) was displayed in (B). Renal microcirculation was determined with a moor image in three IR groups of rats (C). Renal IR significantly decreased renal microcirculation, as indicated by PU value in IR rats vs. IRMV and IRDEXMV treated rats (n = 6 each) (D). Renal PU significantly recovered towards the normal value within 10 min reperfusion in IRMV-and-IRDEXMV treated rats (n = 6 each). (E): The percentage change of blood flow influx mean is shown. Renal arterial blood flow is significantly decreased in IR and IRMV rats, but not in IRDEXMV rats (n = 6 each) (F). Percentage change of renal arterial blood flow is significantly increased in IRMV− and IRDEXMV−treated rats vs. IR rats within 10 min reperfusion (n = 6 each) (G). Renal IR significantly increases blood urea nitrogen (H) and creatinine (I) levels in IR−, IRMV−, and IRDEXMV− treated rats (n = 6 each). However, these two levels are significantly decreased in IRDEXMV vs. IR rats. * p < 0.05 vs. CON rat. MV, microvesicle; IR, ischemia/reperfusion; IRMV, ischemia/reperfusion with microvesicle; IRDEXMR, ischemia/reperfusion with dexmedetomidine preconditioned microvesicle; PU, perfusion unit; ROS, reactive oxygen species. # p < 0.05 vs. IR rat. a p < 0.05 vs. IRMV rat.
As displayed in Figure 3C, in the baseline period, the kidney displayed a red color with high PUs in three groups of rats. Forty-five-minute ischemia severely decreased the renal microcirculation and PUs in IR-, IRMV-, and IRDEXMV-treated kidneys. In contrast, during the reperfusion stage, there was significant recovery of renal microcirculation in the IRMV and IRDEXMV groups vs. the IR group ( Figure 3D). Depressed renal microcirculation by ischemia was significantly recovered towards normal values within 10 min reperfusion in the IRMV and IRDEXMV groups vs. the IR group. The percentage change of blood flow influx mean was displayed in the order of IRDEXMV > IRMV > IR rats ( Figure 3E). We further confirmed renal blood flow response by a transonic flowmeter. Our data observed that post-IR renal arterial blood flow was significantly decreased in IR and IRMV rats, but not in IRDEXMV-treated rats ( Figure 3F). A significant increase in percentage change of renal arterial blood flow within 10 min reperfusion was found in IRMV and IRDEXMV vs. IR kidneys ( Figure 3G). The percentage change of renal arterial blood flow was significantly increased in IRDEXMV as compared to IRMV rats, implying a faster recovery of renal arterial blood flow with DEXMV treatment.
Renal IR significantly increased blood urea nitrogen ( Figure 3H) and creatinine ( Figure 3I) levels in IR-, IRMV-, and IRDEXMV-treated rats. However, these parameters were significantly decreased in IRDEXMV vs. IR rats, implying that IRDEXMV treatment was more efficient in improving renal function than IRMV.

MVs and DEX-MVs Reduced IR-Induced Inflammation and Apoptosis
We found that a significantly increased tubular injury score, including epithelial flattening, tubular atrophy, tubular dilatation and brush border and erythrocyte accumulation, was demonstrated in IR kidneys ( Figure 4A), whereas the tubular injury score was significantly reduced in IRMV and IRDEXMV kidneys ( Figure 4C). Furthermore, IRDEXMV decreased the tubular injury score further than IRMV did.
We explored the effect of MVs on renal IR-evoked inflammatory transcription factor (pNF-κB), apoptosis-related caspase-3, and PARP expression in four groups of rats. Compared to CON, the expression of pNF-κB, c-caspase-3, and PARP expression was all enhanced in IR kidneys ( Figure 4B), whereas the expression in pNF-κB ( Figure 4D), cleaved caspase-3 ( Figure 4E), and PARP ( Figure 4F) was significantly downregulated in IRMV and IRDEXMV kidneys. Our data also implied that DEX preconditioning ADSC MVs were more effective in the IRDEXMV than in the IRMV group at attenuating IR-induced inflammation and apoptosis.

MiR-122-5p Was the Highest-Expression miRNA in IR Kidneys
The differentially expressed miRNAs and the whole distribution of differentially expressed miRNAs were clustered and plotted with a heatmap ( Figure 5A). Compared to CON, a significant upregulation of miR-122-5p expression levels was found in the IR group, while downregulation of miR-122-5p expression levels was found in the IRMV and IRDEXMV groups (p < 0.05, Figure 5B). We further noted that the miR-122-5p expression levels in IRDEXMV were significantly lower than those in the IRMV group. We confirmed the significant upregulation of miR-122-5p expression in IR kidneys vs. CON kidneys by quantitative real-time PCR (qRT-PCR) and significant downregulation of miR-122-5p in the IRMV and IRDEXMV groups ( Figure 5C). The action site varication by TargetScan ( Figure 5D), quantitative PCR ( Figure 5E), and dual luciferase reporter gene assays (Figure

MiR-122-5p Was the Highest-Expression miRNA in IR Kidneys
The differentially expressed miRNAs and the whole distribution of differentially expressed miRNAs were clustered and plotted with a heatmap ( Figure 5A). Compared to CON, a significant upregulation of miR-122-5p expression levels was found in the IR group, while downregulation of miR-122-5p expression levels was found in the IRMV and IRD-EXMV groups (p < 0.05, Figure 5B). We further noted that the miR-122-5p expression levels in IRDEXMV were significantly lower than those in the IRMV group. We confirmed the significant upregulation of miR-122-5p expression in IR kidneys vs. CON kidneys by quantitative real-time PCR (qRT-PCR) and significant downregulation of miR-122-5p in the IRMV and IRDEXMV groups ( Figure 5C). The action site varication by TargetScan ( Figure 5D), quantitative PCR ( Figure 5E), and dual luciferase reporter gene assays ( Figure 5F) were used to confirm that erythropoietin (EPO) was a target of miR-122-5p. Collectively, these findings indicated that IR increased miR-122-5p expression and decreased EPO expression as compared to the CON group. MV treatment significantly reduced miR-122-5p expression and increased EPO expression in the IRMV and IRDEXMV groups. A further downregulation of miR-122-5p expression and an upregulation of EPO expression were found in the IRDEXMV group as compared to the IRMV group.
Antioxidants 2022, 11, x FOR PEER REVIEW 14 of 25 5F) were used to confirm that erythropoietin (EPO) was a target of miR-122-5p. Collectively, these findings indicated that IR increased miR-122-5p expression and decreased EPO expression as compared to the CON group. MV treatment significantly reduced miR-122-5p expression and increased EPO expression in the IRMV and IRDEXMV groups. A further downregulation of miR-122-5p expression and an upregulation of EPO expression were found in the IRDEXMV group as compared to the IRMV group.

MVs and DEX-MVs Decreased IR-Induced Apoptosis by Western Blot
To verify the possible role of miR-122-5p-targeted gene EPO expression, we compared the expression of EPO and Bax/Bcl-2/C-caspase 3/PARP-mediated apoptotic signaling pathway in these four groups of kidneys by Western blot analysis. Figure 6A showed the typical Western blot of five proteins' expression in four groups of rats. Compared to CON kidneys, the significantly upregulated expression of Bax, Bax/Bcl-2 ratio (Figure 6B), c-caspase-3 ( Figure 6C), and PARP ( Figure 6D) was indicated in IR kidneys, whereas the downregulated expression of Bcl-2 was demonstrated in IR kidneys ( Figure 6A). When compared to IR kidneys, the significant decrease in Bax/Bcl-2 ratio, c-capase-3, and PARP expression was found in IRMV and IRDEXMV kidneys.
Because EPO could attenuate DNA fragmentation and prevent caspase-3 activation through upregulation of Bcl-xL [22], we also determined the renal EPO expression in response to IR injury. Renal EPO expression was significantly decreased in IR kidneys vs. CON kidneys, while the depressed EPO expression was partly recovered in IRMVand IRDEXMV-treated kidneys ( Figure 6E). As compared to IR kidneys, the degree of recovered renal EPO expression was significantly enhanced in IRDEXMV kidneys, and not in IRMV kidneys.

MiR-122-5p Inhibitor or EPO Attenuated H/R Induced Annexin V-Apoptosis through Upregulation of Bcl-2 and Downregulation of Bax in NRK-52E Cells by Immunofluorescence
To test the functional role of miR-122-5p and EPO on H/R injury in the renal proximal tubular cells, we transfected miR-122-5p mimics and inhibitor into NRK-52E cells and analyzed the expression level of Bcl-2-, Bax-, and Annexin V-mediated apoptosis by an immunofluorescence assay. As shown in Figure 7A, H/R markedly enhanced Bax and depressed Bcl-2 expression, subsequently leading to the increase in Annexin V-apoptosis expression in NRK-52E cells. MiR-122-5p mimics significantly decreased Bcl-2 expression (p < 0.05, Figure 7B), increased Bax expression (p < 0.05, Figure 7C), and promoted Annexin V-mediated apoptosis (p < 0.05, Figure 7D) in these cells. H/R plus miR-122-5p mimics seem to further decrease Bcl-2 expression and enhance Bax expression, resulting in a higher Annexin V-mediated apoptosis formation in the NRK-52E cells. The use of miR-122-5p inhibitor significantly decreased H/R injury by the upregulated Bcl-2 expression and the downregulated Bax expression, contributing to a lower Annexin V-apoptosis in NRK-52E cells. We verified the possible role of EPO on H/R injury of NRK-52E cells. Our data observed that EPO treatment significantly reduced H/R injury through the upregulation of Bcl-2 expression and downregulation of Bax expression and Annexin V-apoptosis formation.

MiR-122-5p Inhibitor or EPO Ameliorated H/R-Induced Apoptosis through the Upregulation of Bcl-2 and Downregulation of Bax in NRK-52E Cells by Western Blot
To confirm the functional role of miR-122-5p and EPO on H/R injury in the renal proximal tubular cells, we transfected miR-122-5p mimics and inhibitor into NRK-52E cells and analyzed the expression level of Bcl-2-, Bax-, C-caspase 3-, and PARP-mediated apoptosis by Western blot. As shown in Figure 8A, the original Western blot data consistently showed that H/R and/or miR-122-5p mimics markedly decreased EPO and Bcl-2 expression associated with the increased Bax, C-caspase 3, and PARP expression in NRK-52E cells. MiR-122-5p inhibitor significantly increased H/R-depressed EPO expression (p

MiR-122-5p Inhibitor or EPO Ameliorated H/R-Induced Apoptosis through the Upregulation of Bcl-2 and Downregulation of Bax in NRK-52E Cells by Western Blot
To confirm the functional role of miR-122-5p and EPO on H/R injury in the renal proximal tubular cells, we transfected miR-122-5p mimics and inhibitor into NRK-52E cells and analyzed the expression level of Bcl-2-, Bax-, C-caspase 3-, and PARP-mediated apoptosis by Western blot. As shown in Figure 8A, the original Western blot data consistently showed that H/R and/or miR-122-5p mimics markedly decreased EPO and Bcl-2 expression associated with the increased Bax, C-caspase 3, and PARP expression in NRK-52E cells. MiR-122-5p inhibitor significantly increased H/R-depressed EPO expression (p < 0.05, Figure 8B), Bcl-2 expression (p < 0.05, Figure 8D), decreased H/R enhanced Bax expression (p < 0.05, Figure 8C), C-Caspase 3 (p < 0.05, Figure 8E), and PARP expression (p < 0.05, Figure 8F) in NRK-52E cells. The use of EPO significantly decreased H/R injury by the preservation of EPO and Bcl-2 expression and the downregulation of Bax, C-Caspase 3, and PARP expression, subsequently leading to less apoptosis formation in the NRK-52E cells.

Discussion
Several studies utilizing mesenchymal stem cells from bone marrow, adipose tissue, and umbilical cord are ongoing, and cumulated reports of the beneficial effects of cellular therapeutics for renal IR and renal disorders are indicated [23][24][25] through the actions of paracrine effect of stem-cell-released MVs and miRNAs. In the present study, we preconditioned DEX-treated ADSC-MVs to increase more biofactors with higher migrating activity in vitro and improve renal H/R injury in vitro and IR injury in vivo. For example, DEX-treated ADSC-MVs were more efficient than ADSC-MVs at significantly increasing CD44 expression, a positive biomarker of ADSCs [15], and improve IR-depressed renal microcirculation, renal arterial blood flow, and renal function by the inhibition of apoptosis formation. We found that IR promoted the highest miR-122-5p expression among all the miRNAs in the damaged kidneys. In addition, we discovered that miR-122-5p was directly targeted to the EPO gene and downregulated EPO expression, resulting in severe apoptosis signaling in IR kidneys. We further demonstrated that the cell-free, newly, efficiently, and safely therapeutic MV from DEX preconditioning ADSCs containing specific biofactors to inhibit miR-122-5p induced detrimental effects, subsequently ameliorating renal IR injury.
Excess ROS formation contributes to renal IR injury [1,26]. ROS-induced renal proximal tubular apoptosis is a pivotal step in the pathogenesis of acute renal IR injury, which can be rescued by the treatment of antioxidant MnSOD [26]. Although strategies for inhibiting ROS production or increasing their degradation and scavenging have been attempted, the application of MV to prevent or treat renal IR injury by their ROS scavenging activity has not explored. Our present data in Figure 1F displayed that MV derived from ADSCs or DEX-preconditioned ADSC-MVs contained some antioxidant factors to directly scavenge ROS activity in vitro. Further, our in vivo model also demonstrated that both MV and DEX-MV treatments could attenuate renal IR-enhanced superoxide anion activity in the damaged kidney (Figure 2A,B). However, DEX-MVs exerted stronger antioxidant activity than MVs to decrease the ROS amount in vitro and in vivo.
One previous study reported that combined exendin-4 and ADSC therapy was superior to either one alone for improving acute kidney IR injury, suggesting the further protection raised by pharmacological preconditioning [10]. In the present study, we applied anti-inflammatory and renoprotective DEX [11,12] to precondition ADSCs. Our data found that DEX-preconditioned ADSC-MVs significantly reduced miR-122-5p-enhanced oxidative stress, Bax-/caspase 3-/PARP-mediated apoptosis, phosphorylated NF-κB-mediated inflammation, and BUN/creatinine levels in IR kidneys. Stem-cell-derived MVs or exosomes have been identified as a new mechanism of paracrine secretion of stem cells and exploited as a new therapeutic approach of stem cells to ischemic diseases [27,28]. Little is known about the biogenesis and the molecular composition of MVs produced by ADSC in different physiopathological states. Our data observed that the amount of MV protein is increased after appropriate sonication and informed that increased MV amounts could be obtained by the mechanical stimulation, whereas increased biofactors in MVs can be adapted by DEX preconditioning. Cantaluppi et al. [29] implied that MVs derived from endothelial progenitor cells protect the kidney from IR injury by delivering the miRNA cargo, which contributes to the reprogramming of hypoxic resident renal cells to a regenerative program. Our present results first implied that ADSC-MVs or DEX-preconditioned ADSC-MVs exerted renal protection against IR injury on the improvement of depressed renal microcirculation and renal blood flow and renal dysfunction. DEX-preconditioned ADSC-MVs were more efficient than ADSC-MVs in ameliorating renal hemodynamics and dysfunction through the action of downregulating miR 122-5p and negatively upregulating the target gene EPO expression.
IR-evoked excess oxidative stress contributed to endothelial dysfunction and decreased microcirculation and renal vasoconstriction, leading to renal dysfunction [21]. Recent evidence indicated that endothelial cells subjected to H/R injury were dysfunctional and coupled with increased apoptosis and ROS overproduction, and were rescued by endothelial progenitor cell-derived MVs via their carried miRNA-126 and eNOS activity associated with the depressed ROS production and activated PI3K/eNOS/NO pathway [32]. Our data also evidenced that ADSC-MVs or DEX treated ADSC-MVs can decrease ROS production and improve the decreased microcirculation and renal arterial blood flow, suggesting the existing antioxidant and vasodilatory factors in our prepared MVs. Importantly, our data further informed that DEX-MVs were more efficient than MVs to recover renal hemodynamics, indicating the beneficial effect of DEX pretreatment on ADSC.
As with miR-122-5p through the inhibitory action on EPO expression in the IR kidneys, our data first evidenced that upregulation in miR-122-5p could reduce EPO expression by luciferase activity and Western blot. In addition, the functional assay of miR-122-5p on Bax/Bcl-2/apoptosis signaling evidenced that miR-122-5p mimics upregulated Bax and downregulated EPO and Bcl-2 expression, consequently resulting in the increased Annexin V-apoptosis stains in the NRK-52E cells. MiR-122-5p inhibitor treatment significantly downregulated Bax and upregulated EPO and Bcl-2 expression in the NRK-52E cells. Golmohammadi et al. [33] reported that vitamin D3 and EPO protect against renal IR injury via the upregulation of heat shock protein 70 and antiapoptotic microRNA-21 expression. MiRNAs did play an important role in regulation of IR injury. Compared to mesenchymal stem-cell MVs (untreated), the EPO-treated mesenchymal stem-cell MVs had a greater benefit in reduction of apoptosis appearance in mice kidneys with unilateral ureteral obstruction, a chronic kidney disease model [34]. In response to acute kidney injury, EPO intervention can boost up the differentiation function of bone-marrow-derived mesenchymal stem cells and reverse their low secretion effect [35]. It is likely that the changes in the miRNAs' profile and the increased EPO concentration contributed to the decreased levels of apoptosis in the proximal tubular epithelial cells, and improved renal function in kidney diseases. This would be found in our renal IR model. On the other hand, DEX preconditioning alone could ameliorate renal IR injury and inflammatory response through the p38-CD44-pathway and possibly through ischemic preconditioning [36], suggesting DEX's effect on enhancing CD44 expression, which is a positive biomarker of rat ADSCs [15]. Exosomes or MVs of MSC origin have cell-surface markers such as CD29, CD44, and CD73 embedded in them, playing a vital role in the biomechanisms involved in the repair and regeneration, bioenergetics, immunoregulation, intracellular communication, and tissue metabolism [37]. Our data also found that intrarenal arterial DEX-MV treatment significantly increased CD44 expression compared to MV in the kidney ( Figure 2C,D).
The further increased CD44 expression may confer more efficient therapeutic potential in DEX-MV through the enhancement of homing and tissue-regenerating ability.

Conclusions
In conclusion, DEX-preconditioned ADSC-derived MVs can ameliorate IR-induced tubular cell apoptosis and renal dysfunction through downregulated miR-122-5p/Bax/PA-RP/apoptosis signaling and upregulated target-gene EPO expression in the post-IR kidney ( Figure 9). DEX-preconditioned ADSC-derived MVs could be an important tool for utilization during renal IR injury. Furthermore, an actual advantage would come from the therapeutic use of MVs with respect to MSCs, avoiding the possible immune rejection and lung entrapment, improving the safety, and allowing the crossing of biological barriers.
omarker of rat ADSCs [15]. Exosomes or MVs of MSC origin have cell-surface markers such as CD29, CD44, and CD73 embedded in them, playing a vital role in the biomechanisms involved in the repair and regeneration, bioenergetics, immunoregulation, intracellular communication, and tissue metabolism [37]. Our data also found that intrarenal arterial DEX-MV treatment significantly increased CD44 expression compared to MV in the kidney ( Figure 2C,D). The further increased CD44 expression may confer more efficient therapeutic potential in DEX-MV through the enhancement of homing and tissue-regenerating ability.

Conclusions
In conclusion, DEX-preconditioned ADSC-derived MVs can ameliorate IR-induced tubular cell apoptosis and renal dysfunction through downregulated miR-122-5p/Bax/PARP/apoptosis signaling and upregulated target-gene EPO expression in the post-IR kidney (Figure 9). DEX-preconditioned ADSC-derived MVs could be an important tool for utilization during renal IR injury. Furthermore, an actual advantage would come from the therapeutic use of MVs with respect to MSCs, avoiding the possible immune rejection and lung entrapment, improving the safety, and allowing the crossing of biological barriers. Figure 9. The summary diagram. The summary diagram suggests that DEX-preconditioned ADSCderived MVs can provide CD44 homing effect; ameliorate IR-induced renal hemodynamics depression; and reduce renal ROS level, tubular cell inflammation, apoptosis, tubular injury score, and renal dysfunction through downregulated miR-122-5p/Bax/PARP/apoptosis signaling and upregulated target-gene EPO expression in the post-IR kidney. DEX-preconditioned ADSC-derived MVs could be an important tool for utilization during renal IR injury. ADSC, adipose stem cells; AKI, acute kidney injury; DEX, dexmedetomidine; EPO, erythropoietin; MV, microvesicle; I/R, ischemia/reperfusion; PARP, poly-(ADP-ribose)-polymerase; ROS, reactive oxygen species.  . The summary diagram. The summary diagram suggests that DEX-preconditioned ADSCderived MVs can provide CD44 homing effect; ameliorate IR-induced renal hemodynamics depression; and reduce renal ROS level, tubular cell inflammation, apoptosis, tubular injury score, and renal dysfunction through downregulated miR-122-5p/Bax/PARP/apoptosis signaling and upregulated target-gene EPO expression in the post-IR kidney. DEX-preconditioned ADSC-derived MVs could be an important tool for utilization during renal IR injury. ADSC, adipose stem cells; AKI, acute kidney injury; DEX, dexmedetomidine; EPO, erythropoietin; MV, microvesicle; I/R, ischemia/reperfusion; PARP, poly-(ADP-ribose)-polymerase; ROS, reactive oxygen species.


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Features of Peripheral Blood Th-Cell Subset Composition and Serum Cytokine Level in Patients with Activity-Driven Ankylosing Spondylitis

Th cells may exhibit pathological activity depending on the regulatory and functional signals sensed under a wide range of immunopathological conditions, including ankylosing spondylitis (AS). The relationship between Th cells and cytokines is important for diagnoses and for determining treatment. Accordingly, the aim of this study was to investigate the relationship between Th-cell subset composition and serum cytokine profile for patients with activity-driven AS. In our study, patients were divided into two groups according to disease activity: low-activity AS (ASDAS-CRP < 2.1) and high-activity AS (ASDAS-CRP > 2.1). The peripheral blood Th cell subset composition was studied by flow cytometry. Using multiplex analysis, serum cytokine levels were quantified and investigated. It was found that only patients with high-activity AS had reduced central memory (CM) Th1 cells (p = 0.035) but elevated numbers of CM (p = 0.014) and effector memory (EM) Th2 cells (p < 0.001). However, no activity-driven change in the Th17 cell subset composition was observed in AS patients. Moreover, low-AS activity patients had increased numbers of Tfh17 EM cells (p < 0.001), whereas high-AS activity was associated with elevated Tfh2 EM level (p = 0.031). The serum cytokine profiles in AS patients demonstrated that cues stimulating cellular immunity were increased, but patients with high-AS activity reveled increased IL-5 level (p = 0.017). Analyzing the data obtained from AS patients allowed us to conclude that Th cell subset differentiation was mainly affected during the CM stage and characterized the IL-23/IL-17 regulatory axis, whereas increased humoral immunity was observed in the high-AS activity group.

Introduction
Ankylosing spondylitis (AS) is a systemic disorder characterized by chromic injury to the paravertebral tissue, spinal joints and sacroiliac joint, with ankylosing progression occurring in the intervertebral joints and calcification developing in the spinal ligament. Currently, AS pathogenesis has not been fully elucidated. However, genetic predisposition (association with HLA-B27) and activated immune system [1][2][3] have been shown to have associations. The elevated production of some cytokines (particularly IL-17 and IL-23), which stimulate effector immune cells to markedly increase tumor necrosis factor-alpha (TNF-α) overproduction, subsequently inducing a joint inflammatory process, is recognized as one of the key mechanisms initiating this disorder [4][5][6].
Thus, the current study investigates the features of Th-cell subset composition and serum cytokine profiles for patients with activity-driven AS.

Assessing Peripheral Blood Th Cell Subset Composition in Activity-Driven AS Patients
While investigating the peripheral blood Th cell quantities and relevant cell subset compositions in AS patients, it was found that the percentages and absolute numbers of total helper T cells were unrelated to AS activity (Table 1). Assessing the surface CD45RA and CD62L co-expression levels revealed that this repertoire changed with Th cell maturation and the acquisition of effector cell functions, allowing us to identify a 'naïve' Th (CD45RA + CD62L + ) subset and estimate the level of mature as well as central memory (CM), effector memory (EM) (CD45RA -CD62L + and CD45RA -CD62L -, respectively), and terminally-differentiated effector memory CD45RA + T cells (TEMRA, CD45RA + CD62L -) [27,28]. We found that only patients with low-activity AS had lowered percentages of peripheral blood CD45RA -CD62L -EM Th cells. In contrast, high-activity AS was linked to decreased levels of peripheral blood CD45RA + CD62L + 'naïve' Th cells compared to those of the control group, whereas in high-vs. low-activity AS, the levels of CD45RA -CD62L + CM Th cells were elevated. Table 1. Peripheral blood Th-cell subset composition in activity-driven AS patients (Ме (IQR)). We also performed a detailed analysis of CM and EM Th cell composition by assessing the expression of cell-surface chemokine receptors CCR4, CCR6, CXCR3 and CXCR5. It was found that low-activity AS patients vs. the control group had elevated absolute numbers of peripheral blood CM Th, along with increased percentages of CCR6 + Th-cells (Table 2). In contrast, high-activity AS patients were shown to have decreased percentages of CXCR5 -CXCR3 + CCR6 -CCR4 -Th1 cells and elevated levels of CXCR5 -CXCR3 -CCR6 -CCR4 + Th2 cells, as well as CCR6 + Th17 cells, along with increased absolute numbers of CM Th, compared to the control group.

Parameters Control Low-Activity AS
Our investigation of CM Th17 cell subset composition revealed that both low-and high-activity AS had increased percentages of CXCR5 -CXCR3 -CCR6 + CCR4 + 'classical' Th17 and CXCR5 -CXCR3 -CCR6 + CCR4 -DN Th17 cells, along with decreased levels of CXCR5 -CXCR3 + CCR6 + CCR4 + Th17.1 cells (Table 3), compared to the control group. Percentages of CM Th-cell subset composition are presented as proportions of the total CM Th cell population; р 1 -significant difference compared with the control group; р 2 --//-patients with low-activity AS. The CM Tfh cell subset composition in AS patients is shown in Table 4. In particular, it was found that regardless of disease activity, patients had lowered percentages of CXCR5 + CXCR3 + CCR6 -CCR4 -Tfh1 cells, as well as elevated quantities of Tfh2 and Tfh17 cells with CXCR5 + CXCR3 -CCR6 -CCR4 + and CXCR5 + CXCR3 -CCR6 + CCR4phenotypes, respectively, compared to the control group. At the same time, high-and low-activity AS patients were found to have decreased percentages of CXCR5 + CXCR3 + CCR6 + CCR4 + 'double-positive' DP Tfh cells compared to the control group. Table 4. Peripheral blood CM Tfh-cell subset composition (%) in activity-driven AS patients (Ме (IQR)).

CM Tfh Cell Subsets
Control Low-Activity AS High-Activity AS Chemokine receptor expression was also analyzed in EM Th-cells. It was found that low-activity AS patients had elevated percentages of peripheral blood CCR6 + EM Th-cells (Table 5) compared with the control group, whereas high-activity AS was characterized by an increased percentage of CXCR5 -CXCR3 -CCR6 -CCR4 + Th2 cells compared with the low-activity AS and control groups. Our assessment of EM Th17 cell subset composition revealed that regardless of AS activity, patients had increased percentages of 'classical' Th17 and DN Th17 cell subsets (CXCR5 -CXCR3 -CCR6 + CCR4 + and CXCR5 -CXCR3 -CCR6 + CCR4 -, respectively) ( Table 6), compared with the control group. Table 5. Peripheral blood chemokine receptor-based EM Th-cell subset composition in activity-driven AS patients (Ме (C 25 -C 75 )).

Parameters
Control Low-Activity AS High-Activity AS Percentages of EM Tfh cell subsets are presented as proportions of the total EM Th cell population; р 1 -significant difference compared with the control group; р 2 --//-patients with low-activity AS. However, only low-activity AS patients vs were found to have increased percentages of CXCR5 + CXCR3 -CCR6 + CCR4 -Tfh17 cells (Table 7) compared to the control group. Furthermore, a decreased level of CXCR5 + CXCR3 + CCR6 -CCR4 -Tfh1 cells was observed in both AS patient groups compared with the control group. Moreover, in high-activity AS patients, an increased percentage of CXCR5 + CXCR3 -CCR6 -CCR4 + Tfh2 cells, paralleled with a decreased percentage of CXCR5 + CXCR3 + CCR6 + CCR4 + DP Tfh cells, was observed, as compared with control and low-activity AS subjects. Table 7. Peripheral blood EM Tfh-cell subset composition (%) in activity-driven AS (Ме (C 25 -C 75 )).

EM Tfh Cell Subsets
Control Low-Activity AS High-Activity AS

Serum Cytokine Profile in Activity-Driven AS Patients
A multiplex assay allowed us to investigate the serum cytokine profiles of AS patients. It was found that low-activity AS patients had elevated levels of IL-2 and IL-17E/IL-25 (Table 8) compared to the other groups. At the same time, only high-activity AS patients had increased serum levels of IL-15. Regardless of the disease activity, AS patients had increased concentrations of serum IL-12(р40), IL-13, IL-17A, IL-17F, IFN-γ and MSCF, compared to the control group.

Discussion
Developing effector Th-cell subsets undergo a two-stage maturation comprising antigen-independent thymic differentiation and antigen-dependent differentiation occurring in the secondary peripheral immune organs. The first stage results in egress of 'naïve' CD45RA + CD62L + Th cells into peripheral blood, whereas stage 2 involves subsequent antigen-dependent differentiation of the central memory (CM, CD45RA -CD62L + ), effector memory (EM, CD45RA -CD62L -) and terminally-differentiated effector Th cells (TEMRA, CD45RA + CD62L + ) [27,29,30]. T cell differentiation may be assessed by detecting the surface CD45 isoform, a transmembrane tyrosine phosphatase-coupled protein. The extracellular CD45 portion is encoded by seven exons [31][32][33]. 'Naïve' Т cells are found to bear all domains in this region of CD45 molecules, being denoted as CD45RA (with a molecular weight of 220 kDa). During antigen-dependent differentiation, CD45 mRNA undergoes alternative splicing, resulting in the loss of CD45 extracellular domains to be further transformed via intermediate isoforms. TEMRA Th-cells also express CD45RA [34]. Hence, Th-cells which are negative for CD45RA are defined as memory Th-cells (CM and EM), whereas Th cell subsets expressing it are referred to as the naïve and TEMRA subsets.
Another surface molecule being expressed on Th-cells which is used for phenotyping is CD62L, an L-selectin that accounts for cell migration into peripheral lymphoid tissues by recognizing cognate endothelial ligands such as GlyCAM-1 (glycosylation-dependent cell adhesion molecule-1), MadCAM-1 (mucosal addressin cell adhesion molecule-1) and CD34 (endothelial cell protein) [35,36]. Therefore, CD62L is expressed on naïve and CM T cells (accounting for their migration into peripheral lymphoid tissues) but not on EM T cells or TEMRA (to function outside lymphoid tissues) [37].
While analyzing co-expressed the CD45RA and CD62L profiles in Th-cells in AS patients, it was found that low disease activity was associated with a decreased percentage of peripheral blood EM Th-cells, whereas high-activity AS was linked to a lower absolute number of naïve Th-cells but an increased level of CM Th-cells. Overall, it may be concluded that alterations in Th-cell phenotypes during cell differentiation in AS patients reflect the disease course. CM Th-cells are characterized as a long-living cell subset which mainly recirculates via secondary lymphoid tissues [38,39]. In contrast, EM Th-cells are mature effector cell types displaying lowered proliferative potential, even though their capacity to produce and secrete diverse cytokines, along with the expression of effector molecules, substantially exceeds those found in CM Th-cells [39,40].
Next, we conducted a detailed analysis of the CM and EM Th-cell subset composition according to the surface chemokine receptor-based expression for CCR4, CCR6, CXCR3 and CXCR5.
Based on the expression for receptors CCR4, CCR6, CXCR3 and CXCR5 on Thcells, the major polarized peripheral blood helper T cell subsets were determined: Th1-(CXCR5 -CXCR3 + CCR6 -CCR4 -), Th2 (CXCR5 -CXCR3 -CCR6 -CCR4 + ), Th17 (CXCR5 -CCR6 + ) and Tfh cells (CXCR5 + ) [51][52][53][54]. It was found that low-activity AS patients had elevated percentages of Th17-cells, along with an increased absolute number of CM Th cells. At the same time, high-activity AS patients had increased percentages of peripheral blood Th2 and Th17 cells but decreased Th1 cell levels and increased absolute numbers of CM Th cells. Th17 cells are the major source of IL-17 production; they are also able to secrete a broad cytokine and chemokine profile which primarily exerts pro-inflammatory activity. Not surprisingly, a cytokine multiplex assay in AS patient sera revealed elevated concentrations of IL-17 family (IL-17A, IL17E/IL-25 и IL-17F) cytokines; this was virtually unrelated to disease activity. Among them, IL-17A and IL-17F may act on stromal cells and lymphocyte subsets, which initiate inflammatory responses [55,56]. In addition, it was evidenced that IL-17A may mediate bone injury by inducing RANK expression on the osteoclast surface, as well as upregulating RANKL production via mesenchymal stem cells [26]. It is also worth emphasizing that elevated levels of IL-17 cytokine family members were revealed in our therapeutic interventions.
Th1/Th2 balance has been widely investigated in AS immunopathogenesis [6,19,26]. It has been shown that Th1 cells elicit an immune response against intracellular pathogens by secreting IFN-γ, which acts as a macrophage-activating cue [26,57]. Its serum concentration in the patients examined in this study was elevated, regardless of the AS activity. Apart from IFN-γ, Th1-cells are also able to produce IL-2, IL-10 and TNF-α, which are involved in inflammatory processes. However, a single negative correlation was only observed between peripheral blood CM Th1 cells and serum MCSF (macrophage colony-stimulating factor) levels. MCSF (CSF-1), bearing a core of the four α-helices, is the key cytokine, ensuring macrophage proliferation, differentiation and survival; it is produced by fibroblasts, activated macrophages, osteoblasts, activated endothelial cells as well as bone marrow stromal cells [58][59][60]. It was confirmed that osteoblast-released MCSF exerts a paracrine activity on osteoclasts that results in bone resorption [61]. It may be assumed that during AS, such a relationship characterizes a developing competitive interplay between innate and adaptive immunity, which may become evident during therapeutic intervention.
Th2 cells are important for the eradication of extracellular parasites; their cytokine profile consists of IL-4, IL-5 and IL-13 [26,57]. Hence, elevated levels of CM Th2 cells, along with lowered Th1-cell levels in high-activity AS patients, characterize a prevalence of protective type 2 immune responses. In particular, only this cohort of patients featured elevated serum IL-5 levels, which shows a positive association between Th2 cells and serum IL-4 concentration.
Four major Th17 cell subsets were identified within the total CCR6 + CM and EM Th cell population, which differed both in terms of the chemokine receptor CXCR3 and CCR4 expression patterns as well as the range of functional activities [62][63][64]. To date, 'classical' CCR4 + CXCR3 -Th17-cells, double-positive CCR4 + CXCR3 + Th17-cells (DP Th17 cells), nonclassical CCR4 -CXCR3 + or Th17.1-cells as well as double-negative CCR4 -CXCR3 -(DN Th17 cells) subsets have been distinguished. 'Classical' Th17 cells are able to actively produce IL-17A, whereas the release of cues such as IL-22 and GM-CSF is not prominent. We observed that the related cell proliferation was less markedly suppressed by proinflammatory cytokines. Non-classical vs. 'classical' Th17 cells show higher TCR-driven proliferative potential. Th17.1 cells may produce a great quantity of GM-CSF, accounting for neutrophil recruitment and activation in the site of inflammation, whereas IFN-γ elicits tissue macrophage activation [65,66]. Moreover, Dankers et al. (2021) demonstrated that both classical and non-classical Th17 cells, together with synovial fibroblasts, may become mutually activated by generating an inflammatory loop by recruiting other proinflammatory immune cells and releasing matrix metalloproteases [62]. DN Th17 vs. classical Th17 cells expressed higher levels of IFN-γ, IL-17A, IL-17F, MIP-3a/CCL20 and TNF-α, whereas the potential to express the IL-13 encoding gene was reduced. It is believed that it is the CCR6 + DN cells which largely produce IL-17F and IL-8. Moreover, DN Th17 cells express high levels of mRNA for the molecules which are responsible for lymphoid tissue migration (CCR7, CXCR5, CXCL13, SELL, SIRP1, JAM3, AIF1) [66,67]. Among all Th17 cell subsets, DP Th17 cells (CXCR5 -CXCR3 + CCR6 + CCR4 + ) possess the lowest capacity to produce IFN-γ, IL-17A, TNF-α and IL-13. However, they may express adhesion molecules and chemokine receptors, ensuring cell migration into the site of inflammation [62]. We found that regardless of AS activity, the quantity of both peripheral blood CM classical and DN Th17-cells was increased, in parallel with a decline in DP Th17-cell levels. Therefore, even during therapeutic interventions, the AS patient cohort showed increased percentages of actively-cytokine producing Th17 cells, as confirmed by the multiplex assay. With this in mind, low-activity AS was associated with a positive correlation between CM CCR6 + DP cells and serum IL-6 levels that characterize changes in the related cell counts and the relevant activity of inflammatory events. In addition, it is worth noting that cytokine IL-6 stimulates Th17 cell differentiation, resulting in an imbalance between pathogenic Th17 and regulatory T cell subsets [68]. With high disease activity, a correlation between levels of classical Th17 cells and serum IL-4 was observed that further characterizes a more profound involvement of the humoral immune arm in AS immunopathogenesis under high disease activity.
Tfh cells oversee the fully controlled development of B-cell-dependent humoral immune response in germinal center follicles within secondary lymphoid tissues (B cell proliferation, plasma cell and memory B cell differentiation, immunoglobulin isotype switching, etc.). Moreover, Tfh-cells produce quite a broad range of diverse cytokines, among which the major cues are IL-10 and IL-21 [69][70][71]]. An approach based on assessing surface CXCR3 and CCR6 chemokine receptor expression was used to analyze the composition of the Tfh-cell subset [72]. Owing to this, a whole set of human circulating peripheral blood Tfh-cells was divided into four distinct subsets: Tfh1-cells (CXCR3 + CCR6 − ), Tfh2-cells (CXCR3 − CCR6 − ), Tfh17-cells (CXCR3 − CCR6 + ) and dual-positive Tfh-cells (DP Tfh-cells, CXCR3 + CCR6 + ). Tfh1 cells (CXCR5 + CXCR3 + CCR6 -CCR4 -) share some Th1 cell properties, including CXCR3 expression (in the absence of CCR6 surface expression), as well as a potential to produce IFN-γ in response to functional stimuli [73,74]. Furthermore, it was revealed that such Tfh1 cells down-regulate antibody production by 'naïve' B cells in vitro [72]. In contrast, Tfh2 cells have similarities with Th2 cells, in particular, the expression of transcription factor GATA3 and the coupled production of the IL-4, IL-5 and IL-13 cytokines. Therefore, in Tfh2 cells co-cultured with 'naïve' B cells, the latter enhanced production of all major antibody classes [73,75]. Similar to Th17 cells, Tfh17 cells similar to Th17 cells are able to produce IL-17A and IL-22 in parallel with expressing the transcription factor ROR t. Moreover, Tfh17 cells may also elevate in vitro antibody production by naïve B cells except IgE, so that the peak effect may be observed when IgA production is induced [71,76]. DP Tfh-cells are able to extensively produce IL-21 and IL-4 [77]. We found that regardless of AS activity, the level of peripheral blood CM Tfh1 declined in parallel with an increase in the quantity of CM Tfh2 and Tfh17 cells. It should be noted that the dominance of Tfh2 and Tfh17 cells over the Tfh1 cell subset is related to the immunopathogenesis underlying systemic and organ-specific autoimmune disorders [78]. Based on the imbalanced Tfh cell subset composition and subsequent cytokine production, a pivotal role for the immunopathogenic IL-23/IL-17-axis has been identified. This was confirmed by the positive correlation between peripheral blood CM Tfh2 cells and serum IL-12(p40) level found in low-activity AS; its concentration was elevated both in low-and high-activity AS patients. IL-12(p40) is a constituent in both IL-12 and IL-23 cytokines which is also able to act as a macrophage chemoattractant and to stimulate activated dendritic cells [79,80]. Paiva I.A. et al. (2021) noted that it is IL-12(p40) that initiates an IL-23/IL-17 cytokine cascade [81]. In addition, low-activity AS patients had levels of CM Tfh2 cells that were positively correlated with serum IL-5 concentrations which characterize functional activity of this cell type and the manifestation of humoral mechanisms during AS. The cellular arm in AS immunopathogenesis was confirmed by the positive correlation between serum MCSF level and the total CM Tfh (CXCR5 + ) and Tfh17 cell subset levels. At the same time, high-activity AS was linked to a negative correlation between total CM Tfh cells and serum level for IL-6 and IL-17F. It was revealed that amino acid homology between IL-17A and IL-17F comprises 50% but IL-17F is produced at an earlier stage of Th17 cell differentiation [81]. Moreover, it was shown that IL-17A stimulates ongoing immune response 10-30 times more readily than IL-17F [82]. Overall, it may be suggested that the correlation data account for the competitive relationships between the mechanisms underlying cellular and humoral immunity, being more pronouncedly evident in high-activity AS. Furthermore, only patients with high-activity AS were found to have decreased percentages of peripheral blood CM DP Tfh-cells.
While CM Th-cells subsequently differentiate into the EM subset, other features related to an altered Th-cell subset composition in AS patients were revealed. For instance, only low-activity AS was paralleled with a decreased count of total EM Th17 (CCR6 + ) cells, whereas high-activity AS was associated with elevated levels of Th2 cells. Furthermore, high-activity AS was associated with a positive relationship between peripheral blood Th2 cell level and serum IL-4 concentration. Such a relationship has been previously identified for this CM Th cell subset which accounts for humoral immunity and is involved in AS immunopathogenesis.
In investigating the EM Th17 cell subset composition in activity-driven AS patients, we detected similar changes. For instance, regardless of the disease activity, patients had elevated levels of classical and DN Th17 cells in peripheral blood. However, only highactivity AS patients showed a relationship between the Th17 cell subset composition and some serum cytokine levels, corroborating the role played by this cell subset in activating the cellular mechanisms underlying AS immunopathogenesis. In particular, a positive correlation between the level of classical Th17 cells and serum cytokine concentrations for IL-12(p40) and IFN-γ, as well as between total Th17 (CCR6 + ) cells and the DP Th17 subset, together with IFN-γ level, characterize the activation mechanisms for this Th-cell subset and its role in stimulating cell-mediated immunity in AS.
Finally, it is worth noting that the relationships observed for Th17 cells were observed solely in high-activity AS patients. Altogether, it was found that the AS disease course is characterized by an altered peripheral blood Th-cell phenotype in parallel with cell differentiation stage. Associations between certain serum cytokine or chemokine levels and percentage of any of the Th cell subsets examined in this study may suggest that they may be somehow linked, suggesting potential molecular or cellular targets for further interventions to alleviate AS, although this remains to be further investigated.

Patients
The study was conducted at the clinic of the Krasnoyarsk State Medical University from 2019 to 2021. Fifty-eight AS patients (14 females 44 males), aged 20-58 years old (Ме = 40.0 years [32.0; 47.0]), were enrolled. AS was verified based upon the New-York Criteria [83]. The inclusion criteria were disease onset over the age of 18 years and signed informed consent form. The exclusion criteria were as follows: the presence of other types of spondyloarthritis, osteoarthritis, acute and chronic exacerbated diseases; HIV infection; syphilis; immunodeficiency; hematological or oncological diseases; as well as other clinically significant conditions that may affect the results of the study. The inclusion criteria for healthy subjects was the absence of acute and chronic diseases. Disease activity was assessed using the BASDAI and ASDAS indices, adjusted to ESR and CRP levels according to the current nomenclature approved by the Assessment of SpondyloArthritis International Society and Outcome Measures in Rheumatology (ASAS/OMERACT) [84]. Based on disease-driven activity, all patients were stratified into two groups: absence or low activity AS (ASDAS-CRP < 2.1; n = 37) and high activity AS (ASDAS-CRP ≥ 2.1; n = 21). The clinical and laboratory patient characteristics with varying AS activity are shown in Table 9. Twenty-four AS patients received anti-TNFα (adalimumab-6 patients, infliximab-14, etanercept-3, golimumab-1) and 15 received anti-IL17 (secukinumab-8 patients, netakimab-7). All preparations were used at recommended doses and frequency of administration, with bDMARDs length comprising 1.5 [1.0; 4.5] years. Patients receiving standard therapy were treated with non-steroidal antiinflammatory drugs (NSAID)-19 subjects (100%), glucocorticoids-13 subjects (68.4%), sulfasalazine-7 subjects (36.8%) and methotrexate-3 subjects (15.8%). Activity-driven AS patient stratification according to therapy intervention is shown in Table 10. The control group comprised 45 healthy volunteers (15 females and 30 males) aged 18-57 years old (Ме = 39.0 years [27.0; 47.0]). The study protocol was approved by the Ethics Committee at the Krasnoyarsk State Medical University (protocol 87/2018, dated 14/12/2018) and was conducted in accordance with the Declaration of Helsinki. Written consent was obtained from all participants. Table 9. Patient clinical characteristics in activity-driven AS.

Parameters
Low-Activity AS High-Activity AS р Quantitative data are presented by the median and interquartile ranges (median (IQR)). Qualitative variables are presented in absolute values and percentages (n (%)). Comparisons were made using the Mann-Whitney U test (for quantitative variables) and the Fisher's exact test (for qualitative variables). Table 10. Therapy-based patient distribution in activity-driven AS (n (%)). Limitations: Due to the limited number of patients with high activity AS, the formation of the high activity group (ASDAS-CRP ≥ 2.1; n = 21) was problematic and led to significant differences in the numbers within the groups. However, the size of the groups represented the population of patients with AS in terms of age, gender and type of treatment from Krasnoyarsk region of Russian Federation.

Sample Collection
All experiments were performed within ≤6 h after blood collection. Fasting whole peripheral blood from every patient was collected in vacuum test tubes containing with K3-EDTA anti-coagulant. For cytokine measurement, cell-free plasma samples were obtained after whole blood centrifugation at 300 g for 7 min at +4 • C. Samples were then placed in new 1.5 mL tubes and centrifuged again at 300 g for 15 min at +4 • C to remove residual platelets and other blood cells. Finally, each plasma sample was aliquoted and stored at −80 • C until use.

Th Cell Flow Cytometry Immunophenotyping
An amount of 100 µL of whole blood was used for Th cell immunophenotyping by flow cytometry. Blood samples were stained with antibodies against CD3 (clone UCHT1) and CD4 (clone 13B8.2) to separate CD3 + CD4 + T cells into distinct subsets using antibodies against CD45RA (clone 2H4LDH11LDB9 [2H4]) and CD62L (clone DREG56), based on a previously proposed gating strategy for major Th cell subsets [85]. Naïve Th cells were of phenotype CD45RA + CD62L + , whereas phenotypes CD45RA -CD62L + and CD45RA -CD62Lwere identified as central memory (CM) and effector memory (ЕМ) T cells, and terminally-differentiated effector (TEMRA) CD45RA-positive Th cells were determined to be of the CD45RA + CD62Lsubset. CM and EM Th-cell subsets were further analyzed for chemokine receptor expression using additional marker-specific monoclonal antibodies: CCR4 (CD194, clone L291H4), CCR6 (CD196, clone G034E3), CXCR3 (CD183, clone G025H7) and CXCR5 (CD185, clone J252D4). Antibody staining was performed according to the manufacturer's recommendations. A selection of optimal antibody combinations and relevant-antibody-conjugated fluorochrome combinations was performed according to a previously proposed protocol [86]. RBCs were lysed by adding ex tempore

Statistical Analysis
Data samples were analyzed by calculating the median (Ме) and interquartile ranges (IQR), along with the 25th and 75th percentiles (C 25 -C 75 ). Qualitative variables of clinical parameters were presented in absolute numbers and percentages (n (%)). A significance level for quantitative variables was assessed using a non-parametric Mann-Whitney U test. Qualitative variables were compared using the Fisher's exact test. The strength of parameter relationships was assessed by calculating the Spearman rank R correlation coefficients. Statistical analyses were performed with the Statistica 8.0 software package (StatSoft Inc., 2007). were gated on the side scatter/forward scatter plot with a "LY" gate; (c) Th cells were identified from the gate of lymphocytes as "CD3 + CD4 + " (d) and then separated into differentiated subsets using CD45RA and CD62L expression ('naïve' Th were CD45RA + CD62L + ; central memory Th were CD45RA -CD62L + ; effector memory Th were CD45RA -CD62L -and "terminally differentiated CD45RA-positive" Th cells were CD45RA + CD62L -). Histograms (e-h)-expression of CXCR5, CCR6, CCR4 and CXCR3, respectively, by total Th cell population; regions "CXCR5", "CXCR3", "CCR6" and "CCR4" were used as branches for hierarchical tree histograms (i,k). Hierarchical tree histograms were gated on CM and EM Th-(i,k), respectively). The frequency histograms below the trees indicates the relative proportion of cells in each subset that expressed CXCR5, CXCR3, CCR6 and CCR4 within CM and EM Th.

Multiplex Assay
Serum cytokine concentrations were measured with a MILLIPLEX ® MAP Human TH17 Magnetic Bead Panel (Merck, USA) in xMAP-based technology (Luminex, USA), according to the manufacturer's recommendations. Data were collected and analyzed using a Luminex MAGPIX Instrument (Luminex, USA).

Statistical Analysis
Data samples were analyzed by calculating the median (Ме) and interquartile ranges (IQR), along with the 25th and 75th percentiles (С25-С75). Qualitative variables of clinical parameters were presented in absolute numbers and percentages (n (%)). A significance level for quantitative variables was assessed using a non-parametric Mann-Whitney U test. Qualitative variables were compared using the Fisher's exact test. The strength of were gated on the side scatter/forward scatter plot with a "LY" gate; (c) Th cells were identified from the gate of lymphocytes as "CD3 + CD4 + " (d) and then separated into differentiated subsets using CD45RA and CD62L expression ('naïve' Th were CD45RA + CD62L + ; central memory Th were CD45RA -CD62L + ; effector memory Th were CD45RA -CD62Land "terminally differentiated CD45RA-positive" Th cells were CD45RA + CD62L -). Histograms (e-h)-expression of CXCR5, CCR6, CCR4 and CXCR3, respectively, by total Th cell population; regions "CXCR5", "CXCR3", "CCR6" and "CCR4" were used as branches for hierarchical tree histograms (i,k). Hierarchical tree histograms were gated on CM and EM Th-(i,k), respectively). The frequency histograms below the trees indicates the relative proportion of cells in each subset that expressed CXCR5, CXCR3, CCR6 and CCR4 within CM and EM Th.

Conclusions
T helper (Th) cells are among the most numerous T lymphocyte fractions in cellular composition and account for various immunopathological conditions, including AS activity. It was found that patients with low-activity AS had reduced numbers of peripheral blood EM Th cells (CD45RA -CD62L -), whereas high disease activity was associated with decreased "naive" Th (CD45RA + CD62L + ) cells and increased levels of CM Th cells (CD45RA -CD62L + ). By analyzing Th cell surface chemokine receptor expression, we were able to determine the compositions of the CM and EM Th cell subsets. It was found that patients with high disease activity had decreased percentages of CM Th1 cells but increased level of CM Th2 lymphocytes. Only patients with high AS activity were found to have decreased numbers of CM DP Tfh cells, but decreased and increased levels of CM Tfh1 and Tfh2 cells were observed, regardless of the disease activity. Such changes in the CM Th cell subset composition primarily characterize the main AS immunopathogenetic mecha-nisms, which are initiated and enabled via the IL-23/IL-17 regulatory axis. Using multiplex analysis, we found increased serum levels of cytokines which stimulate the activity of cellular immunity (IL-12(p40), IL-17A, IL-17F, IFN-g and MSCF); notably, this did not appear to depend on disease activity. At the same time, in patients with serum high-AS activity, the concentrations of IL-5 were also increased, which accounts for the importance of the mechanisms underlying humoral immunity under the disease course. A correlation analysis between the CM Th cell subset composition and cytokine levels also confirmed the role of humoral immune mechanisms in high-activity AS. Because our examinations of AS patients were carried out during ongoing therapy, it may be assumed that traditional anti-inflammatory therapy and GIBT have a more pronounced effect on the mechanisms of cellular immunity, whereas the humoral immune response was less markedly inhibited. On the other hand, it can be assumed that the modality of AS course can be determined by the involvement of humoral immune reactions.
Subsequent differentiation of CM Th cells into EM Th cells was found to be associated with altered cell subset composition. Patients with high-activity AS had normalized Th1 cell counts but persistently increased level of Th2 lymphocytes, whereas only low disease activity was associated with persistent high levels of Th17 cells. Only in patients with high disease activity were changes in Th2 cell numbers related to altered IL-4 concentrations. In general, it can be concluded that Th cell subset differentiation in AS patients tends to change mainly at the CM Th cell stage; additionally, it is characterized by the IL-23/IL-17 regulatory axis but is associated with increased activity of the humoral immune mechanisms in cases of high-level disease. The presented features of Th cell differentiation characterize the features of immunopathogenesis depending on the activity of AS and should be taken into account when conducting immunotherapy.  Informed Consent Statement: Informed consent was obtained from all the subjects involved in the study.

Data Availability Statement:
The data generated and analyzed during this study are included in this published article. Additional information is available from the corresponding author on reasonable request.